PROJECT SUMMARY/ABSTRACT Our long-term goal is to elucidate the molecular mechanism of the cytoplasmic dynein-dynactin motor complex, and to define the molecular bases of dynein-related diseases in humans. Dynein is the primary vehicle for microtubule minus-end-directed transport in eukaryotic cells. The function and dysfunction of this vital motor and its regulatory proteins contribute to a broad range of cellular functions and human diseases. Despite increasing efforts to define dynein?s functional properties, the molecular mechanisms that govern dynein?s mechanochemistry remain poorly understood. This deficiency largely stems from dynein?s structural complexity. Dynein belongs to the AAA+ class of ATP-hydrolyzing mechanoenzymes that assemble into ring-shaped structures, and therefore, possesses distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.4 MDa) and structure function studies on dynein have been limited until recently by the availability of functional recombinant dynein. Adding to dynein?s complexity, dynein associates with multiple accessory chains and the dynactin complex, all of which are essential for nearly every cellular function of dynein. Mutations in the dynein heavy chain and dynactin's largest subunit, p150glued, which contains dynactin?s putative microtubule-binding domain, cause devastating neurological diseases. However, mechanistic knowledge of dynein?s function?and therefore its dysfunction? is limited compared to kinesin and myosin, which poses a major barrier for the development of targeted therapies. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with mutagenesis and structure-function studies. We will employ S. cerevisiae, insect and human cell-based expression systems to produce stable wildtype and mutant versions of both multiprotein complexes. Using these biochemical tools and multicolor single-molecule fluorescence and optical tweezers methods, we will decipher the molecular mechanism underlying the processive motion of the dynein-dynactin complex and determine how dynactin regulates dynein force generation. This information will provide insights into cellular physiology and identify targets within the dynein-dynactin complex for therapeutic interventions.